Association of Neurotensin Receptor 1 Gene Polymorphisms With Defense Mechanisms in Healthy Chinese

Aims: In the central nerve system, neurotensin (NT), and neurotensin receptor 1 (NTR1) modulate the dopamine system. Gene variations in the dopamine system have been demonstrated to influence certain defense mechanisms, but no studies have investigated possible effect of NTR1 gene polymorphisms in the biological determination of these defenses. The present study therefore examined this link. Methods: In 412 healthy Han Chinese, single nucleotide polymorphisms rs6090453C/G, rs6011914C/G, and rs2427422A/G of the NTR1 gene were genotyped, and the defense mechanisms were measured by the self-reporting Defense Style Questionnaire 88. Results: Significant male-specific differences in the projective identification among the rs6090453 genotypes (p = 0.003); in the intermediate defense, reaction formation, and projective identification among the rs6011914 genotypes (p = 0.011, 0.010, and 0.011, respectively); and in the projective identification among the rs2427422 genotypes (p = 0.005) were found when the level of significance was adjusted by the Bonferroni correction. There was no significant difference in any of the defense scores among genotypes of any single nucleotide polymorphism in the total cohort or female subjects (all p > 0.017). The distributions of genotypes between the low and high score subgroups showed significant differences in the rs2427422 genotype distributions for help-rejecting complaining, regression, and projective identification (p = 0.010, 0.022, and 0.044, respectively). Significant differences were found between males and females in 10 defense mechanisms (all P < 0.05). Conclusions: The gene variations in the NTR1 polymorphisms were involved in the biological mechanisms of intermediate defense mechanisms, and this effect was influenced by sex.


INTRODUCTION
The concept of the psychological defense mechanism is one of the cores in the psychoanalytic theory, which was first proposed by Freud (1). With the establishment and development of the personality structure model (id, ego, and superego) in the psychoanalytic school, the concept of the defense mechanism changed constantly (2). In the Diagnostic and Statistical Manual of Mental Disorders-Fourth Edition, defense mechanisms were defined as "automatic psychological processes that protect the individual against anxiety and from the awareness of internal or external dangers or stressors. Individuals are often unaware of these processes as they operate" (3). Psychological defense mechanisms represent a crucial component of individual capacity to maintain emotional homeostasis. Without them the conscious mind would be much more vulnerable to negatively charged emotional input, such as that pertaining to anxiety and sadness (4,5).
In fact, until defense-related scales were developed, it had always been difficult to study defense mechanisms since they always worked at the unconscious level. Bond et al. developed and revised the self-reporting Defense Style Questionnaire 88 (DSQ-88) to assess possible conscious derivatives of defense mechanisms and to elicit manifestations of a subject's characteristic style of dealing with conflict, either consciously or unconsciously, based on the assumption that the subject was able to accurately comment on his/her behavior from a distance (6). The authors provided evidence for testing the reliability of the DSQ-88 by assessing psychiatric patients and healthy subjects (7)(8)(9). Since then, this questionnaire has been applied to a large body of clinical research (10)(11)(12)(13)(14)(15)(16).
The relationship of defense mechanisms with the psychopathology of mental disorders and abnormal behaviors is not been clearly understood, and in cases conflicting (17). Nevertheless, most studies have indicated strong evidence that adaptation of defense style correlated with mental health and that some diagnoses were correlated with specific defense patterns, for example, patients with depression or personality disorders tended to use more maladaptive defenses and less adaptive defenses while patients with anxiety disorders in general tended to use more immature and neurotic defenses (18,19). Therefore, it is important and necessary to elucidate the determination of defenses.
There have been many previous studies on the social psychological factors of defense mechanisms in psychology; however, few have focused on biological factors. Moreover, the majority of previous studies focused on psychoanalytic and not statistical investigations (2). Using the data from twin-based studies, Andrews conducted a multivariate genetic analysis of three factors, including trait anxiety, locus of control and defense style, and demonstrated that defense style was substantially influenced by genetic factors and there was a significant loading (0.44) on a common genetic factor contributing to the variance of individual defenses (20). Therefore, biogenetic factors play a role in the determination of defense mechanisms. It is known that defense mechanisms are an important and enduring facet of personality (8) and that the dopamine system is closely related to personality traits (21)(22)(23)(24). It was thus speculated that the dopamine system was associated with defenses. Two studies confirmed this hypothesis: Coming et al. demonstrated that the dopamine D2 receptor gene (DRD2) locus was the site of one of the factors that control defenses (25) and Huang et al. demonstrated that the PPP1R1B gene, encoding the dopamineand cAMP-regulated phosphoprotein (DARPP-32), was one of the factors that was responsible for defenses (26).
In the central nervous system, neurotensin (NT) is a 13-amino acid multifunctional neurotransmitter and neuromodulator. Among the three NT receptor (NTR) subtypes, NTR1 and NTR2 are G protein-coupled receptors, and NTR1 has a much higher affinity for NT than NTR2 (27,28). The NTR1 gene is located on the 20q13 locus and consists of four exons and three introns (29). A large amount of anatomical, physiological, pharmacological and behavioral evidence has demonstrated that NT transmission modulates central dopaminergic functions (30)(31)(32)(33). Therefore, the genes of the NT system may contribute to defense mechanisms by regulating dopamine neurotransmission. Moreover, our team has already demonstrated an association between NTR1 gene single nucleotide polymorphisms (SNPs) and personality traits assessed by the Tridimensional Personality Questionnaire (TPQ) in Chinese Han subjects (22,34). Therefore, we decided to test the hypothesis that the NTR1 SNPs were associated with defenses.
To our knowledge, none of the previous reports have found an association between NTR1 gene polymorphisms and defense mechanisms measured by DSQ. In this study, we investigated whether genetic variants in the NTR1 gene were associated with defenses in a large healthy Chinese Han population, and then analyzed any association by sex. The results would provide empirical evidence for the biological factors of defense mechanism. The three SNPs investigated (rs6090453, rs6011914, and rs2427422, Figure 1) were selected due to our previous research on personality traits (22,34), copying styles (35), anxiety (36), schizophrenia (37), and alcohol dependence (38).

Subjects
All subjects were unrelated healthy Chinese-Han volunteers (n = 412, 196 males, 216 females) without psychiatric, neurological, or chronic physical illnesses. The age range was 19-58 years, while the mean ± standard deviation (SD) was 31.43 ± 8.16 years. There was no significant difference in age between the males and females (p = 0.187; Supplementary Table 1). The volunteers were recruited from healthy undergraduate and graduate students and staff of China Medical University, Shenyang, Liaoning Province, China, as well as healthy individuals undergoing physical examinations at the First Affiliated Hospital of China Medical University. Written informed consent was obtained from all participants. All the protocols in the present study were approved by the Ethics Committee of China Medical University (number of ethical approval: 2019-209-2). FIGURE 1 | Genomic structure of NTR1 gene. The NTR1 gene has 4 exons (E) and 3 introns, whose sizes are indicated. The rs6090453C/G, rs6011914C/G, and rs2427422A/G polymorphisms of the NTR1 gene are located in intron 1, whose locations are indicated.  Bond's DSQ-88 was translated and validated into the Chinese version in 1993, with no differences in meaning or content to that of the original version. The Chinese version of DSQ-88 evaluates 24 defense styles, which are classified into three factors of defense: immature or maladaptive (projection, passive aggression, acting out, help-rejecting complaining, fantasy, splitting, regression, and somatization), intermediate or neurotic (reaction formation, undoing, inhibition, withdrawal, idealization, pseudo-altruism, omnipotence, isolation, projective identification, denial, affiliation, consumption, and anticipation), and mature or adaptive (sublimation, suppression, and humor). All participants were asked to complete the questionnaire by themselves within 40 min and to check that all items had been scored. Following the completion of the questionnaire, 2 mL of venous blood from each participant was obtained for genotyping. Each defense style score was represented by the average of all  the items representing the particular defense mechanism, while each defense factor score was derived from the mean of all the defense style scores belonging to the defense factor. According to the study of Bond et al. (6), a subject was considered to score high on a particular defense style and use it if his score was 0.5 SD above the mean on the particular defense. A cutting point of 0.5 SD provided the best discrimination between those who use a defense style and those who do not (6). Thus, subjects could be divided into low score (non-use) and high score (use) subgroups for each defense style based on whether the scores were above or below the cutting point.

Polymorphism Genotyping
Amplification of gene fragments containing the SNPs rs6090453, rs6011914, and rs2427422 by polymerase chain reaction and subsequent identification of genotypes by restriction fragment length polymorphism analysis were carried out as described in our previous studies (22,(34)(35)(36)(37)(38).

Statistical Analysis
Data are presented as the mean ± SD, frequency, or percentage. Data statistical analyses were conducted using SPSS R version 17.0 software (SPSS Inc., Chicago, IL, USA). Potential genotypic associations of the three SNPs with DSQ-scores were detected by one-way analysis of variance (ANOVA) or by the nonparametric Kruskal-Wallis test, depending on homogeneity or heterogeneity of variance of data. Separate analysis by gender was also carried out. The Hardy-Weinberg equilibrium (HWE) and the differences in genotype frequencies between the low and high score subgroups were assessed by chi-square test. The differences in defense mechanisms between male and female subjects were compared by independent sample t-test. Linkage

HWE Results
The numbers of subjects with rs6090453 genotype CC, CG, and GG were 43, 177, and 192, respectively.

LD and Haplotype Analysis Results
As shown in The frequencies of haplotypes composed of the alleles in the three SNPs were shown in Table 1. The haplotype blocks with low frequencies (<1%) were rejected. The two most frequent  haplotypes were GGG (0.659) and CCA (0.243). None of the frequencies of these five haplotypes differed significantly between the male and female samples (all p > 0.05).

Effects of NTR1 Gene Polymorphisms on DSQ Scores in the Total Cohort and in Male and Female Subjects
As shown in Table 2, all DSQ dimension scores among three genotypes of each SNP were compared. There was a marginal difference only in the score of help-rejecting complaining among the three genotypes of the rs2427422 polymorphism (F = 3.028, p = 0.050), but this difference was not significant when the level of significance was adjusted using Bonferroni correction [α ′ = α/k, where α is the unadjusted significance level for pair-wise comparisons, k is the number of independent significance tests, and α ′ (0.05/3 = 0.017) is the corrected significance level for multiple comparisons]. The differences in all DSQ dimension scores among the genotypes of the three polymorphisms were then analyzed separately by sex. We demonstrated significant male-specific differences in the intermediate defense, reaction formation, projective identification, affiliation, mature defense, sublimation, and suppression scores among the rs6090453 genotypes (F = 3.445, 3.131, 5.982, 4.023, 3.552, 3.892, and 3.061, respectively; p = 0.034, 0.046, 0.003, 0.019, 0.031, 0.022, and 0.049, respectively); in the intermediate defense, reaction formation, undoing, and projective identification scores among the rs6011914 genotypes (F = 4.651, 4.760, 3.999, and 4.631, respectively; p = 0.011, 0.010, 0.020, and 0.011, respectively); and in the projective identification score among the rs2427422 genotypes (F = 5.433, p = 0.005; Table 3). Moreover, the male-specific differences in the projective identification score among the rs6090453 genotypes, in the intermediate defense, reaction formation, and projective identification scores among the rs6011914 genotypes, and in the projective identification score among the rs2427422 genotypes were still significant (all p < 0.017) when the level of significance was adjusted by the Bonferroni correction. At the same time, we demonstrated significant female-specific difference in the projective identification score among between the rs6090453 genotypes (F = 3.580, p = 0.030), and in the passive aggression, intermediated defense, and affiliation scores among the rs6011914 genotypes (F = 3.775, 3.080, and 3.273, respectively; p = 0.024, 0.048, and 0.040, respectively; Table 4). However, all these female-specific differences in defense scores among the genotypes of the three SNPs were not significant when the level of significance was adjusted by the Bonferroni correction.

Comparison of the Genotype Distributions Between the High and Low Score Subgroups
As shown in Table 5, the genotypes of the three SNPs between the high and low score subgroups for three defense factors and 24 defense styles were compared. We found significant differences in the rs2427422 genotype distributions between two subgroups for help-rejecting complaining, regression, and projective identification (χ 2 = 9.182, 7.665, and 6.236, p = 0.010, 0.022, and 0.044, respectively).

Comparison of DSQ Scores Between Male and Female Subjects
When the DSQ scores were compared between male and female subjects, significant differences were found in 10 defense styles including fantasy, regression, inhibition, withdrawal, omnipotence, isolation, projective identification, sublimation, suppression, and humor ( Table 6, all p < 0.05). The suppression score for males was higher than that for females, while the scores of other nine defense styles were higher for females.

DISCUSSION
The main finding of the present study was that NTR1 gene SNPs were significantly associated with two particular defense styles (reaction formation and projective identification) and intermediate defense factors to which these two defense styles belong, in healthy Han-Chinese subjects, and that this association was specific to males. On the part of an association between the NTR1 gene and DSQ-measured defenses, any relevant research reports were not be found before the present study.
Defense mechanisms have been considered to result from the interaction of biological and environmental factors (20,26), but the research about the biogenetic basis for defenses has been rarely studied. Generally, defense mechanisms are considered to be an enduring and important dimension of personality and not just an epiphenomena of psychopathology (8). Many previous studies have demonstrated a significant  (26). Gene variants in the dopamine system might thus be associated with defense mechanisms. Previous studies have concluded strong evidence for a regulatory role of NT in the dopamine system. Animal and human radioimmunoassay studies have found that NT was present in all the mammalian brain structures containing dopamine nerve cell bodies and terminals (30,47). Moreover, NTR1 localized both pre-synaptically and post-synaptically at dopaminergic synapses expressing DRD2 (48). The anatomical overlap between NT, NTR1, dopamine, and DRD2 suggests possible interactions that regulate neurophysiological functions at the cellular level. Numerous studies have demonstrated that NT could regulate the affinity of DRs for dopamine and dopamine receptor agonists (30,32,(49)(50)(51), in addition to its action on the excitability of dopaminergic neurons (33,(52)(53)(54). In the present study, we demonstrated that three NTR1 gene polymorphisms were significantly associated with projective identification and that the rs6011914 polymorphism was significantly associated with reaction formation in healthy Han-Chinese males. We also demonstrated significant differences in genotype distributions of the rs2427422 polymorphism between the low and high score subgroups for three defense styles: helprejecting complaining, regression, and projective identification. These results were consistent with and further confirmed the previous study conclusion about the significant association between gene variants in the dopamine system and defense mechanisms (25,26).
Bond reviewed the published studies about the relationships of defense styles with psychopathology and change and proposed that defense styles should become more adaptive with improvement in symptoms, but intermediate defenses tended to become stable over time (19). Recently, Hayden et al. also demonstrated that maladaptive defense mechanisms were significantly reduced during inpatient therapy and remained low until follow-up, whereas neurotic (intermediate) and adaptive defense mechanisms did not change significantly (5). In other words, intermediate defenses have been found to be a trait-like factor among the three defense factors. In a previous study, our team uncovered a particular defense style (undoing) of intermediate defense, which was demonstrated to be associated with FYN gene polymorphisms (55). In the present study, both reaction formation and projective identification, which were demonstrated to be significantly associated with NTR1 gene polymorphisms, were also classified to be intermediate defenses. Therefore, these results might account, at least partially, for the stability of intermediate defenses, and also indicate that relatively stable biogenetic factors including the dopamine system played an important role in the determination of these intermediate defenses.
Moreover, the present study found significant differences among different genotypes in six particular defense styles and intermediate and mature defenses in males, and in three particular defense styles and intermediate defense in females. Interestingly, after the Bonferroni correction, only the differences in the projective identification score among the rs6090453 genotypes; in the intermediate defense, reaction formation, and projective identification scores among the rs6011914 genotypes; and in the projective identification score among the rs2427422 genotypes were still significant in males. The association between NTR1 gene polymorphisms and defenses was thus a sex-specific result. In fact, several previous studies have demonstrated significant differences in defenses between the two genders (56)(57)(58), and in agreement, we found significant differences in 10 particular defense styles between male and female subjects in the present study. Moreover, the important effect of the interaction between gender and gene polymorphisms has already been demonstrated on individual differences in personality traits (59)(60)(61). Therefore, it was expected that our findings were mainly restricted to men. In the present study, we have demonstrated that no significant difference in the frequencies of five haplotypes composed of the alleles in the three NTR1 SNPs existed between the males and females, indicating no sex-specific difference in the haplotype frequency of NTR1 SNPs. Therefore, the present sexspecific findings may be the result of androgens, which might be involved in the effect of the NTR1 gene on neurotransmitter systems, and thus ultimately affect the biological determination of defenses. Biochemical experiments will be needed to determine if this conjecture is indeed true.

CONCLUSION
We investigated the associations between three NTR1 polymorphisms and defense mechanisms measured by DSQ-88 in a large healthy Chinese Han population to demonstrate possible biogenetic mechanisms affecting defenses. We found significant associations in male subjects between three SNPs and the defense style of projective identification as well as the NTR1 rs6011914 polymorphism and the defense style of reaction formation. Therefore, our results provide evidence that gene variants in the NT system can influence the formation and development of defense mechanisms by regulating the dopamine system and that this effect is affected by gender. However, this conclusion should be considered cautiously because of the limited sample size and number of studied SNPs, and the use of a self-rating scale. Further studies of these NTR1 SNPs, as well as other SNPs associated with the dopamine system in larger Chinese Han and other ethnic populations are needed to verify our results and to gain a more comprehensive understanding of the effects of NTR1 allelic variants on defenses.

DATA AVAILABILITY STATEMENT
The original contributions presented in the study are included in the article/Supplementary Material, further inquiries can be directed to the corresponding author/s.

ETHICS STATEMENT
The studies involving human participants were reviewed and approved by the Ethics Committee of China Medical University. The patients/participants provided their written informed consent to participate in this study.

AUTHOR CONTRIBUTIONS
HM and GZ: conceptualization. HM and ML: methodology. HM and JT: software, formal analysis, data curation, and visualization. ML and LZ: validation. HM and LZ: investigation. GZ: resources, supervision, project administration, and funding acquisition. HM and ML: writing-original draft preparation. HM, ML, JT, and GZ: writing-review and editing. All authors contributed to the article and approved the submitted version.